The present invention relates to novel compositions of matter and methods for the treatment of respiratory distress with these novel compositions. This invention also relates to the use of polypeptides (protein fragments) which enhance the surfactant-like properties of phospholipids. More specifically, the present invention relates to novel polypeptides comprising fragment replicas and analogs of fragment replicas of the naturally occurring low molecular weight hydrophobic surfactant associated protein known as SP-B and to their use in the formulation of novel medicaments useful in the establishment, modification and/or maintenance of pulmonary surface tension.
Incorporated herein by reference is U.S. Pat. No. 4,659,805 which discloses and claims a high molecular weight surfactant protein known as SP-A.
The prior art discloses the discovery, method of isolation, characterization and use of a family of naturally occurring mammalian surfactant-associated proteins. Members of this family have been designated as SP-A, SP-B and SP-C. These proteins re known to have the capacity to effect the surfactant-like activity of both natural and synthetic phospholipids. It should be noted that the associated scientific literature also uses the nomenclature of SAP-B, SAP-(Phe), SAP-6 (Phe), and SPL-(Phe) for SP-B. SP-C is also referred to as SAP-C, SAP-(Val), SAP-6 -(Val) and SPL (Val) in the prior art. These two proteins (SP-B and SP-C) are distinct gene products with unique amino acid sequences. Both proteins are derived from proteolytic processing of larger precursor proteins synthesized by pulmonary type II epithelial cells.
SP-B is generated by cleavage of the precursor protein at a glutamine-phenylalanine peptide bond resulting in the naturally occurring protein having 78 amino acid residues, with an N-terminal residue of phenylalanine and a simple molecular weight of about 8,700. SP-B isolated from human lung migrates on polyacrylamide gels as an entity having a relative molecular weight (M.sub.r) of 7-8,000 after sulfhydryl reduction. Without sulfhydryl reduction the naturally occurring protein is also found as large oligiomers. SP-B is extremely hydrophobic, a physical property which is consistent with its in vivo strong association with phospholipids and solubility in organic solvents such as chloroform and methanol.
SP-C has an amino terminal glycine residue, a molecular weight of about 3,700, a polyvaline sequence, and, like SP-B, is also extremely hydrophobic. In addition, both proteins (SP-B and SP-C) are substantially resistant to enzyme degradation by proteases (trypsin, chymotrypsin and staphylococcus nucleotide V-8), endoglcosidase F, and collegenase. Neither SP-B nor SP-C exhibits any degradation or alteration in their molecular weight distribution following treatment with these enzymes. In this behavior, as well as on the basis of amino acid sequence information, the proteins are clearly different from the more hydrophilic and higher molecular weight protein SP-A (also known as SAP-35).
SP-A is present in natural lung surfactant material and has a reduced molecular weight of 30-36,000. SP-A is a glycoprotein containing an internal collgen-like region which is rich in glycine and hydroxyproline. This protein has a N-linked complex carbohydrate and a calcium binding site in the C-terminal globular domain. SP-A is known to bind to phospholipids and is thought to confer important structural organization to the surfactant lipids. This protein is also believed to play a role in preventing the inhibition of pulmonary surfactant activity by plasma or other proteins.
The complete amino acid sequence of SP-B and SP-C has been determined from amino acid analysis and deduced from cDNA's derived from the mRNA's encoding the proteins. The SP-B and SP-C proteins are available as isolates from natural sources, such as bronchioalveolar lung washes and minced lung tissue or as products resulting from the application of recombinant DNA methodologies. When formulated with phospholipids (including synthetic phospholipids) these proteins provide compositions useful in the treatment of pulmonary disorders.
As is often the case with biologically active substances, the isolation of substantial quantities of hydrophobic SP-B and SP-C proteins from natural sources is expensive and labor intensive. Likewise, production of these proteins by recombinant DNA techniques requires substantial effort in terms of design and achieving optimal host/vector expression systems to facilitate production of the proteins. In addition, considerable effort is required to develop effective isolation strategies to separate and purify the expressed protein of interest from the unwanted material. With respect to the specific case of SP-B, the low molecular weight, extreme hydrophobicity and large number of cysteine residues markedly complicates commercial development of efficient expression and/or isolation procedures.
Due to these problems commercial production of SP-B via isolation from natural materials or expression of the protein via recombinant DNA strategies is difficult. The medical community has a need from commercial quantities of SP-B and the present invention fulfills that need through the discovery that only a portion of the SP-B protein molecule is required for the formulation of an effective pulmonary surfactant.
The usefulness of the naturally occurring SP-B and SP-C proteins resides in their ability to significantly improve the surface tension lowering capacity and respreadability of phospholipid admixtures. Natural SP-B and SP-C have been shown, both individually as well as in combination, to facilitate this improvement in surfactant-like activity of phospholipids. However, what has not previously been established is whether the entire protein sequence is necessary to achieve this optimum condition or whether only certain regions or fragments of these proteins, either alone or in certain combinations, might achieve the same result. The prior art fails to suggest, disclose or contemplate the instant discovery. Further one skilled in the art can not a priori determine what fragments will evidence utility or that certain fragments will have activity exceeding that of the complete natural protein.
It is clear that synthesis of replica fragments, or analog thereof, would provide numerous advantages over the chemical or recombinant synthesis of the entire sequence. These advantages include cost, ease of production, isolation and purification.